JP2004150828A - Kinetics analysis method for protein or peptide and substrate for analysis - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for evaluating, and a substrate for evaluating, interaction kinetics of biomolecules or biomolecule aggregates contained in a sample with protein or peptide immobilized on a surface while keeping their activity and function. <P>SOLUTION: This method for evaluating interaction kinetics is characterized in that a plurality of proteins or peptides are immobilized on the same planar substrate, and the interaction kinetics between the biomolecules or biomolecule aggregates contained in the sample and the proteins or the peptides is measured by bringing the proteins or the peptides immobilized on the same planar substrate into contact successively or simultaneously with the sample containing the biomolecules or biomolecule aggregates. <P>COPYRIGHT: (C)2004,JPO

Description

【００３３】
【発明の効果】
本発明により、蛋白質もしくはペプチドを活性・機能を保ったまま表面に固定化し、サンプルに含まれる生体分子もしくは生物分子集合体との相互作用Ｋｉｎｅｔｉｃｓの評価を可能にすることができる。
【図面の簡単な説明】
【図１】Ｄｉｔｈｉｏｂｉｓの構造式
【図２】ＨｉｓＴＡＰＭｙｃとＨｉｓＴＡＰＦｌａｇを固定化したパターン
【図３】ＨｉｓＴＡＰＭｙｃを固定化した部位のシグナル変化（実施例）
【図４】抗Ｍｙｃ抗体、抗Ｆｌａｇ抗体のアフィニティカーブ（実施例）
【図５】抗Ｆｌａｇ抗体を流す前後の像の差
【図６】ＨｉｓＴＡＰＭｙｃを固定化した部位のシグナル変化（比較例１）
【図７】ＨｉｓＴＡＰＭｙｃを固定化した部位のシグナル変化（比較例２）
【図８】抗Ｍｙｃ抗体、抗Ｆｌａｇ抗体のアフィニティカーブ（比較例２）
【図９】実施例で用いたフローセルの概略図（断面）[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a Kinetics analysis method and an analysis substrate that can evaluate an interaction between substances.
[0002]
[Prior art]
In recent years, attempts have been made to examine the function of a protein or peptide biomolecule or to evaluate the interaction between a protein and a biomolecule as a means for clarifying an expressed protein. One known method is to immobilize the protein on the substrate surface and evaluate whether the sample binds to the immobilized site. As a method for detecting binding, a method of labeling fluorescence, radioisotope, or the like is generally used. However, real-time measurement is very complicated, and thus is not suitable for Kinetics measurement for determining a binding rate, a dissociation rate, or a reaction rate. .
[0003]
For example, an assay method in which a peptide is immobilized on a solid surface to evaluate protein interaction has been described. However, since a label molecule is mainly used, the purpose is to detect whether or not the protein is bound (for example, And Patent Document 1). However, in the evaluation of the interaction, the equilibrium constant in the equilibrium state alone is not sufficient, and the Kinetics measurement for evaluating the velocity has become increasingly important in recent years.
[0004]
As a method for measuring Kinetics in real time, a method using surface plasmon resonance (SPR) is known. SPR is a method of detecting a change in the refractive index on a metal thin film. When a substance is bonded, the SPR resonance angle changes. However, normal SPR detects only one point and cannot detect a plurality of points at the same time. Therefore, it is hardly used for screening like a DNA chip.
[0005]
The U.S. patent discloses a method for preparing a biomolecule array on a metal surface and performing interaction analysis at multiple points by SPR imaging, but the purpose is to confirm the presence or absence of binding. The purpose is not to determine Kinetics for calculating (see, for example, Patent Document 2).
[0006]
The classical method for immobilizing proteins and peptides on the surface uses functional groups of proteins and peptides. As a typical technique, there is a method of introducing an acyl azide group, a succinimide group, an epoxy group, or an aldehyde group into the surface and reacting the surface with an amino group of a protein or peptide. However, many functional groups are present in proteins and peptides, and may be immobilized on the surface through functional groups present in sites that control the activity and function of proteins and peptides. In that case, the activity and function as proteins and peptides are lost at the stage of immobilization on the surface, and it is not possible to evaluate the interaction analysis.
[0007]
As a method for immobilizing a protein or peptide while maintaining its activity and function, a method for immobilizing a protein or peptide having a histidine tag with nickel chelate has been proposed. This method can often immobilize proteins and peptides on the surface while maintaining the activity and function. A method of immobilizing a (poly) peptide on a surface with a metal chelate and studying the interaction by SPR has been disclosed (for example, see Patent Document 3). However, here, a method of regenerating and using a chip for each (poly) peptide is used, and the concept of immobilizing a plurality of proteins and peptides on the same surface to measure Kinetics is not shown. .
[0008]
[Patent Document 1]
WO 00/54046 pamphlet [Patent Document 2]
US Pat. No. 6,127,129 [Patent Document 3]
Japanese Unexamined Patent Publication No. Hei 10-505910
INDUSTRIAL APPLICABILITY The present invention immobilizes a protein or peptide having a histidine tag on a surface while maintaining its activity and function, and enables evaluation of Kinetics interacting with a biomolecule or a biomolecule assembly contained in a sample.
[0010]
[Problems to be solved by the invention]
An object of the present invention is to provide a method and a substrate for evaluating a protein or peptide immobilized on a surface while maintaining activity and function, and for evaluating Kinetics interacting with a biomolecule or a biomolecular assembly contained in a sample. Is to do.
[0011]
[Means for Solving the Problems]
As a result of intensive studies, the present inventors have found that the above problems can be solved by the following means.
1. By immobilizing a plurality of proteins or peptides on the same planar substrate, and simultaneously or sequentially contacting the protein or peptide immobilized on the same planar substrate with a sample containing a biomolecule or a biomolecule assembly A method for measuring the interaction Kinetics, comprising measuring the interaction Kinetics between a biomolecule or a biomolecule aggregate contained in the sample and the protein or the peptide.
2. The method for measuring interaction Kinetics as described above, wherein a plurality of proteins or peptides immobilized on a flat substrate have a histidine tag.
3. 3. The method for measuring interaction Kinetics according to 1 or 2, wherein the substrate is a transparent substrate having a gold thin film.
4. 4. The interaction Kinetics according to any one of the above 1 to 3, wherein the interaction Kinetics of a plurality of immobilized proteins or peptides and biomolecules or biomolecule aggregates contained in the sample is measured by surface plasmon resonance. Measuring method.
5. The method for measuring interaction Kinetics according to any one of the above items 1 to 4, wherein the interaction Kinetics is measured by surface plasmon resonance imaging.
6. A substrate characterized in that a thiol compound or a disulfide compound having a nitrilotriacetic acid (NTA) group is immobilized on a gold thin film on a transparent substrate so that a plurality of proteins or peptides having a histidine tag can be immobilized.
7. 7. The substrate according to the above item 6, wherein a thiol compound or a disulfide compound having an NTA group and a thiol compound or a disulfide compound having no NTA group are mixed and immobilized.
8. 8. The substrate according to the above item 6 or 7, wherein the region having a large amount of NTA groups is surrounded by a region containing almost no NTA groups, and nine or more regions having a large amount of NTA groups are discontinuously provided.
9. 9. An array, wherein a plurality of proteins or peptides having a histidine tag are immobilized on the substrate according to any one of the above items 6 to 8 by NTA-Ni chelate.
10. The method for measuring interaction Kinetics according to any one of the above items 1 to 5, wherein the array according to the above item 9 is used as a substrate.
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in detail. The present invention discloses a method for simultaneously or sequentially measuring the interaction Kinetics of a plurality of proteins or peptides, and a substrate used for the measurement.
[0013]
As a method for measuring the activity or function of a protein or peptide, a method of immobilizing the protein or peptide on a substrate and analyzing the interaction with a sample containing a substance to be measured has been considered. In particular, the importance of Kinetics measurement for determining the rates of binding, dissociation, reaction, etc., has recently become increasingly important.
[0014]
No particular limitation is imposed on the method of immobilizing it on the surface, as long as the activity and function are not easily lost. Classically, an amine coupling method and the like can be mentioned, but the activity / function may be lost due to surface immobilization. A method that has recently attracted attention is to introduce a tag amino acid sequence or peptide / protein into a protein or peptide and immobilize it on the surface via the tag. Tags have recently become popular as a means of purifying expressed proteins. In the present invention, it is preferable to fix to the substrate surface via the tag.
[0015]
Examples of the tag include a histidine tag, glutathione-S-transferase, and TAP. Among them, the histidine tag has an advantage that not only can it be easily introduced into a protein or a peptide, but also can be immobilized firmly while maintaining its activity. Is preferred for the purpose of
[0016]
The histidine tag is introduced at the N-terminus or C-terminus of the protein or peptide sequence, and may be introduced together with other tags. A normal histidine tag has a sequence of six histidines and is chelated with a nitrilotriacetic acid (NTA) group-nickel.
[0017]
As a means to perform Kinetics measurement to determine the rate of binding or dissociation, if the binding or dissociation of the protein or peptide immobilized on the substrate with the biomolecule or biomolecule aggregate in the sample can be measured in real time Although not particularly limited, it is preferable because surface plasmon resonance (SPR) can be measured in real time and in a label-free manner, and an SPR imaging method capable of capturing a plane reflection image uses a chip in which a plurality of proteins or peptides are immobilized in an array. It is particularly preferable because it can be analyzed.
[0018]
Hereinafter, a measurement method by the SPR imaging method will be described as an example.
Since SPR is an optical detection method, the substrate used for measurement needs to be transparent.
In addition, a metal thin film is formed on one surface of the transparent substrate to excite surface plasmon resonance. This metal thin film is preferably gold. The gold thin film is not only suitable for SPR measurement, but also a thiol compound or a disulfide compound can be immobilized on a gold (1,1,1) surface by using a gold-sulfur bond. It is preferable that the gold thin film is formed by depositing chromium of 1 nm and gold of 45 nm by vapor deposition because there is no unevenness in the surface thickness and the gold thin film is hard to peel off.
[0019]
The method of introducing an NTA group is preferably a method in which a thiol compound or a disulfide compound having an NTA group is dissolved in ethanol, and a gold surface is immersed to form a self-assembled surface of the thiol compound or the disulfide compound. Since the NTA group is sterically large and has steric hindrance, a method in which a thiol compound or disulfide compound having an NTA group and a thiol compound or disulfide compound having no NTA group are mixed, dissolved in ethanol, and bound to a gold surface. Is particularly preferred. As the thiol compound or disulfide compound having no NTA group, a nonionic substance is preferable in order to suppress nonspecific adsorption of a substance contained in the sample. For example, an alkane thiol having an OH group, an alkane thiol having a polyethylene glycol, and the like can be given. However, it is preferable that the length of the alkane chain of the nonionic alkanethiol or nonionic disulfide is equal to that of the thiol compound or the disulfide compound having the NTA group. If either of them is long, the hydrophobic van der Waals force of the longer compound is strong, and a self-organized surface in which both are mixed may not be obtained.
[0020]
Since the surface thus obtained has substantially no ionic functional groups other than the NTA group, correct Kinetics can be obtained. If the sample substantially has an ionic functional group other than the NTA group, the ionic binding force or repulsion of the measurement target in the sample affects Kinetics, and correct data may not be obtained. Here, “having substantially no” means having or not having a significant influence on Kinetics measurement (two significant figures as a guide), and having or having an absolute amount. Is not determined by
[0021]
Further, by mixing a thiol compound or a disulfide compound having an NTA group with a nonionic thiol compound or a disulfide compound and immobilizing the mixture on the surface, a defect on the self-organized surface formed by the thiol compound or the disulfide compound having an NTA group is obtained. Since the nonionic substance adsorbs and blocks the portion, nonspecific adsorption can be suppressed.
[0022]
In order to measure the interaction Kinetics of a plurality of proteins or peptides, the substrates are preferably arrayed. Although a method of spotting a plurality of proteins or peptides by introducing an NTA group into the entire surface is also possible, it is not preferable because ionic non-specific adsorption occurs on the NTA group in the background where no protein is immobilized. Therefore, an array in which the region into which the NTA group is introduced is surrounded by a region containing almost no NTA group is particularly preferable. The method for manufacturing the array is not particularly limited, but can be performed by a photolithography method or a stamp method.
[0023]
In the present invention, in order to measure the interaction Kinetics of a plurality of proteins or peptides, the substrate preferably has nine or more discontinuous NTA group-introduced regions. This is because Kinetics measurement of many proteins or peptides can be performed simultaneously or sequentially. The upper limit of the number of discontinuous regions is not particularly limited, but is 2500 or less from a practical point of view.
[0024]
The protein or peptide is immobilized on the substrate thus obtained, and observation by SPR imaging is performed while simultaneously or sequentially contacting a sample containing a biomolecule or a biomolecular assembly on the substrate. According to this method, accurate Kinetics can be evaluated by analyzing in real time the reflected light intensity of the site where the protein or peptide is immobilized.
[0025]
The method for simultaneously or sequentially contacting a sample containing a biomolecule or a biomolecule aggregate on a substrate is not particularly limited as long as the sample solution can be brought into contact with the substrate, Considering the ease of observation by the SPR imaging method, it is possible to use a flow cell equipped with a flow channel into which a sample solution flows onto the substrate, switch the contact solution such as a washing buffer and various samples, and observe the change in real time. Is preferred.
[0026]
【Example】
Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited to Examples.
[0027]
[Example]
After depositing 1 nm of chromium on a transparent glass substrate made of SF10 having a thickness of 1 mm and 18 mm × 18 mm, gold was deposited to a thickness of 45 nm. The thickness of the evaporation was monitored with a crystal oscillator. The substrate on which gold was deposited was immersed in a 1 mM ethanol solution of 8-Amino-1-Octanethiol, Hydrochloride (8-AOT, manufactured by Dojindo Laboratories) for 16 hours to form a self-assembled surface of 8-AOT. Was. Succinimide group-terminated polyethylene glycol having a molecular weight of 2,000 (mPEG-SPA, manufactured by Sheawater Polymers) was dissolved in a phosphate buffer at 10 mg / ml, reacted with 8-AOT on the gold surface for 2 hours, and PEG was immobilized on the surface. . A photomask in which a pattern was drawn with chromium on quartz was placed on the substrate, and irradiation was performed with a 1000 W xenon arc lamp manufactured by Oriel for one hour to perform patterning. The pattern of the photomask is one in which 100 squares of 500 μm × 500 μm are arranged. After irradiation, the plate was washed with Milli-Q water and ethanol, and then immersed in an ethanol solution of 475 μM Dithiobis (manufactured by Dojindo Laboratories) and 25 μM mercaptoethanol (manufactured by Nacalai Tesque) for 5 hours. Ethanol was mixed and immobilized. Dithiobis is a disulfide having NTA groups at both ends, and the structural formula is shown in FIG. The biochip thus obtained has an NTA group at the immobilization site, and the background is immobilized with polyethylene glycol, which is a hydrophilic polymer.
[0028]
This biochip was immersed in a 2.5 mM nickel (II) chloride phosphate buffer solution for 1 hour to form an NTA-Ni chelate, and then a histidine-containing protein (HisTAPMyc) into which a Myc peptide was introduced and a Flag peptide were introduced. Escherichia coli cell lysate expressing the histidine-added protein (HisTAPFlag) was diluted 100-fold and spotted at 0.1 μL on the immobilized site. The protein used here is TAP described in Nature Biotech, 1999, 17, 1030-1032, and a histidine tag and Myc or Flag peptide were introduced into TAP. HisTAPMyc and HisTAPFlag were spotted according to the pattern shown in FIG. 2, and after 30 minutes, set on an SPR imaging device (SPRImager: manufactured by GWC instruments). The observation was set in the flow cell schematically shown in FIG. 9 and performed in a buffer of 10 mM Hepes, 150 mM NaCl, pH 7.2. A 0.1 μg / ml anti-Flag antibody (manufactured by Sigma) was first flowed into the flow cell, a buffer was flowed, and then a 0.1 μg / ml anti-Myc antibody (manufactured by Santa Cruz) was flown. The observation was performed at a flow rate of the buffer and the sample of 100 μl / min. FIG. 3 shows a signal change at the site where HisTAPMyc was immobilized. The signal was not increased by the anti-Flag antibody, but was increased only by the anti-Myc antibody. That is, it shows that there is no non-specific adsorption, which is very good. FIG. 4 is a diagram in which the affinity curves of the anti-Myc antibody against HisTAPMyc and the anti-Flag antibody against HisTAPFlag in this case are superimposed. Table 1 shows Kinetics data obtained from this curve. From these results, it was revealed that Kinetics behavior in which Flag adsorbs 5 times faster and desorbs 2 times faster.
In addition, FIG. 5 shows the result of taking a photograph before and after flowing the anti-Flag antibody, and taking a difference between the images before and after flowing. It was possible to observe that the anti-Flag antibody selectively bound to the Flag peptide.
[0029]
[Comparative Example 1]
In the same manner as in the example, a self-assembled surface of 8-AOT was formed on a gold-evaporated transparent substrate, PEG was immobilized, and patterning was performed by ultraviolet irradiation using a photomask. The patterned substrate was immersed in a 500 μM Dithiobis ethanol solution for 5 hours, and Dithiobis was immobilized on a portion irradiated with light. The chip was immersed in a phosphate buffer solution of 2.5 mM nickel (II) chloride for 1 hour, and then the Escherichia coli cell lysate expressing HisTAPMyc and HisTAPFlag was diluted 100-fold, and 0.1 μL was added to the immobilization site. Spotted and set on an SPR imaging instrument. A 0.1 μg / ml anti-Flag antibody was first flowed into the flow cell, a buffer was flowed, and then a 0.1 μg / ml anti-Myc antibody was flowed. FIG. 6 shows a signal change in the portion where HisTAPMyc was immobilized in that case. The anti-Flag antibody tended to non-specifically adsorb to the Myc immobilization site. No correct Kinetics data can be observed on this surface.
It is considered that the reason for this is that Dithiobis alone has a defect in the self-assembled surface, and the anti-Flag antibody is adsorbed on the portion where gold is exposed. In the examples, the mixture was fixed with mercaptoethanol and immobilized, and it is presumed that the defective portion of Dithiobios was filled with mercaptoethanol and nonspecific adsorption was suppressed.
[0030]
[Comparative Example 2]
In the same manner as in the example, a self-assembled surface of 8-AOT was formed on a gold-evaporated transparent substrate, PEG was immobilized, and patterning was performed by ultraviolet irradiation using a photomask. The patterned substrate was immersed in a 1 mM ethanol solution of 7-Carboxy-1-Heptanethiol (7-CHT, manufactured by Dojindo Laboratories) for 5 hours to form a self-assembled surface of 7-CHT on the irradiated portion. . Next, 0.2 M water-soluble carbodiimide (manufactured by Nacalai Tesque) and 0.05 M N-hydroxysuccinimide (manufactured by Nacalai Tesque) are dissolved in a phosphate buffer and reacted for 15 minutes to activate the carboxyl group of 7-CHT. It was made. Immediately, 1 mM AB-NTA (manufactured by Dojindo Laboratories) was reacted for 30 minutes to introduce an NTA group into the immobilization site. Unreacted succinimide groups were blocked by reacting with a 1 M ethanolamine solution (pH 8.5) for 10 minutes.
[0031]
This chip was immersed in a phosphate buffer solution of 2.5 mM nickel (II) chloride for 1 hour, and then the Escherichia coli cell lysate expressing HisTAPMyc and HisTAPFlag was diluted 100-fold, and 0.1 μL was added to the immobilization site. Spotted and set on an SPR imaging instrument. A 0.1 μg / ml anti-Flag antibody was first flowed into the flow cell, a buffer was flowed, and then a 0.1 μg / ml anti-Myc antibody was flowed. FIG. 7 shows a signal change in the portion where HisTAPMyc was immobilized in that case. The anti-Flag antibody tended to slightly nonspecifically adsorb to the Myc immobilization site. Although better than Comparative Example 1, it is inferior to Examples. FIG. 8 shows a diagram in which the affinity curves of the anti-Myc antibody against HisTAPMyc and the anti-Flag antibody against HisTAPFlag are superimposed in this case. These results did not make it possible to clarify the difference in Kinetics behavior.
This is because 7-CHT, which serves as a base for immobilizing AB-NTA, has a negative charge. Not all carboxyl groups of 7-CHT react with AB-NTA, and it is considered that carboxyl groups remain at high density. It can be inferred that Kinetics data is affected because the remaining carboxyl group and the substance in the sample have an ionic interaction. In the case of the examples, since the NTA group is directly immobilized on the gold surface, the ionic effect on Kinetics is small.
[0032]
[Table 1]

[0033]
【The invention's effect】
According to the present invention, it is possible to immobilize a protein or peptide on a surface while maintaining its activity and function, and to evaluate Kinetics interacting with a biomolecule or a biomolecule assembly contained in a sample.
[Brief description of the drawings]
1 is a structural formula of Dithiobis. FIG. 2 is a pattern in which HisTAPMyc and HisTAPFlag are immobilized. FIG. 3 is a signal change in a site in which HisTAPMyc is immobilized (Example).
FIG. 4 Affinity curves of anti-Myc antibody and anti-Flag antibody (Example)
FIG. 5: Difference in image before and after flowing anti-Flag antibody. FIG. 6: Signal change at site where HisTAPMyc is immobilized (Comparative Example 1).
FIG. 7 shows a signal change at a site where HisTAPMyc is immobilized (Comparative Example 2).
FIG. 8: Affinity curves of anti-Myc antibody and anti-Flag antibody (Comparative Example 2)
FIG. 9 is a schematic diagram (cross section) of a flow cell used in an example.

Claims (10)

By immobilizing a plurality of proteins or peptides on the same planar substrate, and simultaneously or sequentially contacting the protein or peptide immobilized on the same planar substrate with a sample containing a biomolecule or a biomolecule assembly A method for measuring the interaction Kinetics, which comprises measuring the interaction Kinetics between a biomolecule or a biomolecule aggregate contained in the sample and the protein or the peptide. The method for measuring interaction Kinetics according to claim 1, wherein the plurality of proteins or peptides immobilized on the flat substrate have a histidine tag. 3. The method according to claim 1, wherein the substrate is a transparent substrate having a gold thin film. The interaction Kinetics according to any one of claims 1 to 3, wherein the interaction Kinetics of a plurality of immobilized proteins or peptides and a biomolecule or a biomolecular assembly contained in the sample is measured by surface plasmon resonance. Measurement method. The method for measuring interaction Kinetics according to any one of claims 1 to 4, wherein the interaction Kinetics is measured by surface plasmon resonance imaging. A substrate characterized in that a thiol compound or disulfide compound having a nitrilotriacetic acid (NTA) group is immobilized on a gold thin film on a transparent substrate so that a plurality of proteins or peptides having a histidine tag can be immobilized. 7. The substrate according to claim 6, wherein a thiol compound or a disulfide compound having an NTA group and a thiol compound or a disulfide compound having no NTA group are mixed and immobilized. 8. The substrate according to claim 6, wherein the region having a large amount of NTA groups is surrounded by a region containing substantially no NTA groups, and the region having a large number of NTA groups is nine or more discontinuously. An array, wherein a plurality of proteins or peptides having a histidine tag are immobilized on the substrate according to any one of claims 6 to 8 by NTA-Ni chelate. The method for measuring interaction Kinetics according to any one of claims 1 to 5, wherein the array according to claim 9 is used as a substrate.

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